Laboratory of Structural & Supramolecular Chemistry

Research

C. Structure of macromolecules by X-ray crystallography

X-ray crystallography allows  to “look” at the detailed structure of biological macromolecules in three dimensions at the atomic level. This is essential in order to understand the macromolecules’ function (or malfunction in case of disease), to propose reaction mechanisms or design drugs. Current and past projects include:

 I.   Binding of γcyclodextrin to Glucogen Phosphorylase b

Selective binding of γ-cyclodextrin to Glucogen Phosphorylase b (GPb) at the glucogen storage site of the enzyme (Fig. C1) [1]. In order to investigate whether the storage site can be exploited as target for modulating hepatic glucose production, αβ and γcyclodextrins (CDs) were identified as moderate mixed-type inhibitors of GPb ( Ki values 47.1, 14.1 and 7.4 mM for αβ and γ-CD, respectively), the γ-CD being better than the others.The structures of GPb with β– and γ-CDs provide an understanding of the binding, which is analogous to this of linear maltopentaose (G5) and maltoheptaose (G7) oligomers namely, anchoring of two glucose residues to GPb. However, the inhibition constant of γ-CD is almost 7-times higher than that of G7 and of β –CD even higher. The lower potency of γ-CD compared to G7 is explained by lack of H-bonds between the γ-CD residues that are next to the anchoring residues and the protein, due to its round shape.

Fig. C1. γCD (in red) binds to the storage site of the Glucose Phosphorylase b dimer.

II.   Ferredoxins 

Structural and electrochemical studies of the new family of 2[4Fe4S] ferredoxins (Fds) from selected pathogenic bacteria such as  Escherichia coli (EcFd), Pseudomonas aeruginosa (PaFd), Allochromatium vinosum (AlvinFd, Fig. C2) in order to correlate their structure to the widely different reduction potentials of their two metal clusters [–460 (cluster II) and –675 mV (cluster I)] [2,3]. The degree of exposure to the outer environment of cluster I sulfur atoms (Fig. C3) leads towards less negative reduction potentials (E°). This is better illustrated by V13G AlvinFd (high exposure, E° = 594 mV) and EcFd (low exposure, E° = 675 mV). In C57A AlvinFd (Fig. C3a), the movement of the protein backbone, as a result of replacing the non-coordinating Cys57 by Ala, leads to a +50 mV up-shift of the potential of the nearby cluster I, by removal of polar interactions involving the thiolate group and adjustment of the H-bonds network involving the cluster atoms.However, the accurate new structures we have determined for the mutated Alvin Fd, C57A and V13G (at 1.05 and 1.48 Å resolution [3], respectively) indicate the effects of subtle structural differences around cluster IIthat provide insight on the electrochemical properties. Namely, polar interactions of side chains and water molecules with the sulfur atoms of cluster II  (Fig. C2), which are absent in the environment of cluster I (the cluster with the lowest reduction potential), can be correlated to the ca. 180-250 mV difference between the reduction potentials of clusters I and II.

Fig. C2. The structure of the Alvin Fd variant, C57A (1.05 Å resolution, a). The environment of cluster II, although  buried inside the molecule, is comparable to the exposed cluster II of the widely studied clostridia ferredoxin (b) that has two isopotential clusters (ca. –400 mV) . This is due to a water molecule (W6) entrapped in the interior of the C57A that attracts side chains of polar residues close to the cluster II S-atoms. In contrast, cluster I of C57A is less polar than cluster II of AlvinFd and of clostridia ferredoxins.

Fig. C3Solvent-accessibility surfaces color-mapped according to the chemical nature of the surface atoms of a, C57A; b, V13G; c, PaFd; d,  EcFd: Nitrogen, oxygen, and carbon atoms are shown in blue, red, and green, respectively. The Cys11SG atoms of cluster I are shown in yellow at the central region of the surfaces. The exposed S1 atom of cluster I of V13G (b) is shown in magenta. The bulky sulfur atom of Met13 of EcFd (d), in orange, limits the exposure of the Cys11SG atom.

III.  Muscle proteins

Collaboration with the group of M. Wilmanns, EMBL-Hamburg on two muscle proteins titin and myomesin

 1.   The structure of the amino terminus titin/telethonin complex [4]: Titin (the largest protein made by human cells) along with myosin and actin, plays a crucial role in the muscle function. The structure of the 2:1 titin:telethonin complex gives the details of the interaction of two titin molecules with telethonin, a smallprotein-component of the the Z-disc of muscles (Fig. C4). This leads to a model of titin interaction in the Z-disc: two titin molecules from different sarcomeric filaments cross-linked at their amino terminus via telethonin. The study may lead to new insight for some muscle and cardiac diseases; moreover, it may provide a molecular paradigm about the cross-linking of major sarcomeric filaments.

Fig. C4. A: ribbon representation (in two orientations) of the two antiparallel titin immunoglobulin-like domains Z1 (blue, residues 1-98) and Z2 (cyan, residues 99-196 including the Z1-Z2 linker) cross-linked by telethonin (red) via extended antiparallel β-sheets involving the three molecules. B: surface representation of the titin-telethonin-titin complex, in two orientations (in green, the telethonin domain 60-90 not participating in β-sheets).

  2. The myomesin structure. The mechanical forces exerted by the muscles are sensed and buffered by unusually long and elastic filament proteins with highly repetitive domain structures. Myomesin is one such repetitive filament protein, which is thought to form bridges between the main contractile filament, that provides the muscle with resistance in the radial dimension. To investigate how the repetitive structure of myomesin contributes to muscle elasticity, the overall architecture has been determined  using a combination of four complementary structural biology methods [5].  The structure of the myomesin domains my9-my11 has been elucidated by X-ray crystallography. Each domain comprises a immunoglobulin-like and helix pattern (Fig. C5 in green). These have been combined with the already available structure of the final my12-my13 domains of the carboxyl terminus in order to make a model of how the myomesin filament attaches in the M-band of the muscle sarcomer.

Fig. C5. The complete myomesin (My) tail-to-tail filament structure: In ribbon representation the dimeric myomesin IgH domain array My9–My10–My11–My12–(My13)2–My12–My11’– My10’–My9′. Myomesin domains that have been structurally investigated are shown in violet (first molecule) and blue (second molecule). The helical linkers are shown in green. A ruler provides an overall length estimate of the filament.

IV.    Endoplasmic Reticulum Aminopeptidases

Endoplasmic Reticulum (ER) aminopeptidases ERAP1 and ERAP2 cooperate to trim a vast variety of antigenic peptide precursors to generate mature epitopes for binding onto MHC class I molecules and help regulate the adaptive immune response.  In collaboration with the group of E. Stratikos at our institution, whave determined for the 1st time the structure of ERAP2 to 3.08 Å by X-ray crystallography [6]. The ERAP2 structure (Fig. C6) provides a structural explanation for the different peptide N-terminus specificities between ERAP1 and ERAP2 and suggests that such differences extend throughout the whole peptide sequence. Overall, the structure helps explain how two homologous aminopeptidases cooperate to process a large variety of sequences, a key property to their biological role. Common coding single nucleotide polymorphisms (SNPs) in ERAP1 and ERAP2 have been linked with predisposition to human diseases ranging from viral and bacterial infections to autoimmunity and cancer. The common ERAP2 SNP rs2549782 that codes for amino acid variation N392K leads to alterations in both the activity and the specificity of the enzyme. Specifically, the 392N allele excises hydrophobic N-terminal residues from epitope precursors up to 165-fold faster compared to the 392K allele, although both alleles are very similar in excising positively charged N-terminal amino acids. This is primarily due to changes in the catalytic turnover rate (kcat) and not in the affinity for the substrate.  X-ray crystallographic analysis of the ERAP2 392K (Fig. C6)allele suggests that the polymorphism interferes with the stabilization of the N-terminus of the peptide both directly and indirectly through interactions with key residues participating in catalysis [7]. The study provides mechanistic insight to the association of this ERAP2 polymorphism with disease and support the idea that polymorphic variation in antigen processing enzymes constitutes a component of immune response variability in humans.

Fig. C6. Left: Ribbon representation of ERAP2  colored by domain (domain I in blue, II in green, III in orange and IV in magenta). Right: A,sequence alignment of ERAP2 and homologous aminopeptidases showing conservation of the polymorphic residue 392 (in bold); the adjacent aminopeptidase motif (HELAH) is also indicated; B, key catalytic residues in the ERAP2K structure are shown in stick representation and superimposed with equivalent residues in ERAP2N (2SE6). Note the relative positioning of Lys392 with respect to the catalytic Glu residues as well as the N-terminus of the bound ligand (Lysa). Electron density 2|Fo|-|Fcat 2σ is indicated around Lys392; C, pairwise electrostatic interaction energies (ΔΕinter in Kcal/mol) calculated between the polymorphic residue 392 and the Lysligand. The reported values are the average of the 20 runs with a standard deviation of less than 1%, which represents the uncertainty due to the granularity of the grid; D, superimposition of ERAP2 residues that cap the S1 specificity pocket of the enzyme. |Fo|-|Fc| electron density (at 2.5σcalculated in the absence of the ligand is shown around Lysa. 2|Fo|-|Fc| electron density at 2σ is indicated around Glu177.

V.    RNA Complexes

Structure determination of complexes of the ribosomal decoding aminoacyl site (A-site) of bacterial 16SrRNA constructs with synthetic analogs of aminoglycosides. Aminoglycoside antibiotics selectively bind to the A-site of bacterial 16SrRNA, interfering in the fidelity of proteosynthesis in the bacterial cellsThe designed compounds are synthesised at the collaborating organic Chemical Biology laboratory of our institution headed by Dr D. Vourloumis. Elucidation of the structure of the above compounds in complex with selective RNA constructs that are appropriate models of the A-site, provides the prerequisite for guiding this synthetic effort. Moreover, the structural information combined with biochemical and kinetic experiments is the first step in the development of pharmaceuticals for fighting bacterial infections by RNA-directed therapies.

Fig. C7. Model of the structure of a ligand-free bacterial RNA construct. Three consecutive molecules of RNA monomers (each represented by a different colour) form infinite continuous chains. The structure has been solved by the software IL MILIONE (Burla MC, Caliandro R, Camalli M, Carrozzini B, Cascarano GL, De Caro L, Giacovazzo C, Polidori G, Siliqi, D, Spagna R, J Appl CRYSTALLOG. 2007, 40   609-613)

VI. DsbD: a bacterial thiol-disulfide oxidoreductase

DsbD is one of the five proteins of the bacterial Disulfide bond (Dsb) formation system. It is located in the inner membrane of Gram-negative bacteria and it is responsible for transporting reducing power from the cytoplasm to the oxidising periplasm of the cell (Fig. C8). It comprises three domains; the central transmembrane domain (tmDsbD), of unknown structure, is flanked by two globular periplasmic domains, the N-terminal domain nDsbD) with an immunoglobulin-like fold and the C-terminal domain (cDsbD) with a classical thioredoxin (Trx) fold. DsbD plays an important role in oxidative protein folding because it allows for the correct formation of disulfide bonds in proteins functioning in harsh extracytoplasmic environments [8]. It is also involved in bacterial pathogenesis as the expression and stability of most virulent factors (secreted molecules, secretion apparatuses, adhesion systems etc) are dependent on the presence of DsbD. The structures of the N-terminal domain in the reduced form and of two point-mutants of the C-terminal domain have been determined [9,10] in collaboration with the Department of Biochemistry, University of Oxford (Prof. Stuart J. Ferguson, Prof. Christina Redfield and Dr Despoina A.I. Mavridou). This work contributed significantly in elucidating the interaction of the soluble domains of this unique oxidoreductase but also in the general understanding of the factors controlling the reactivity of the ubiquitous thioredoxin fold.

DsbD

Fig. C8. DsbD: The sole reductant provider in the periplasm.

VI. Macromolecular crystallization methods

New crystallisation methodology for biological macromolecules, in collaboration with Imperial CollegeLondon.

1. As Coordinators of the Industry-Academia Partnerships and Pathways – Marie Curie Project TOPCRYST, we developed the use of Dual Polarization Interferometry (Fig. C9), pioneered by Farfield Scientific Ltd., to probe crystallisation at its most crucial stages. This allows to predict the outcome of crystallisation trials when they are still at their earliest stages and thus to rationally design and direct such experiments in order to lead them to well-diffracting crystals [11]. Research on other techniques for effective a priori prediction of crystallisation conditions, such as the use of Genetic Algorithms and of calorimetry, is also being actively pursued [12].

DPI

Fig. C9. Optical principle of dual polarization interferometry. Light from a sensing waveguide in contact with the investigated solution interferes with that from a reference waveguide, resulting in interference fringes characterized by their phase and contrast.

2. Research into substances and materials promoting the heterogeneous nucleation of macromolecular crystals is a blossoming topic in crystallogenesis. We have participated in developing the use of materials containing pores of cavities, such as Bioglass, carbon nanotubes, and Molecularly Imprinted Polymers (Fig. C10), as heterogeneous nucleants and are pursuing further ideas [13-16].

crystals from nucleants

Fig. C10. Crystals of a protein (human macrophage Migration Inhibiting Factor) growing at metastable conditions in the presence of Molecularly Imprinted Polymer imprinted with protein

References

Laboratory of Structural & Supramolecular Chemistry

Research

C. Structure of macromolecules by X-ray crystallography

X-ray crystallography allows  to “look” at the detailed structure of biological macromolecules in three dimensions at the atomic level. This is essential in order to understand the macromolecules’ function (or malfunction in case of disease), to propose reaction mechanisms or design drugs. Current and past projects include:

 I.   Binding of γcyclodextrin to Glucogen Phosphorylase b

Selective binding of γ-cyclodextrin to Glucogen Phosphorylase b (GPb) at the glucogen storage site of the enzyme (Fig. C1) [1]. In order to investigate whether the storage site can be exploited as target for modulating hepatic glucose production, αβ and γcyclodextrins (CDs) were identified as moderate mixed-type inhibitors of GPb ( Ki values 47.1, 14.1 and 7.4 mM for αβ and γ-CD, respectively), the γ-CD being better than the others.The structures of GPb with β– and γ-CDs provide an understanding of the binding, which is analogous to this of linear maltopentaose (G5) and maltoheptaose (G7) oligomers namely, anchoring of two glucose residues to GPb. However, the inhibition constant of γ-CD is almost 7-times higher than that of G7 and of β –CD even higher. The lower potency of γ-CD compared to G7 is explained by lack of H-bonds between the γ-CD residues that are next to the anchoring residues and the protein, due to its round shape.

Fig. C1. γCD (in red) binds to the storage site of the Glucose Phosphorylase b dimer.

II.   Ferredoxins 

Structural and electrochemical studies of the new family of 2[4Fe4S] ferredoxins (Fds) from selected pathogenic bacteria such as  Escherichia coli (EcFd), Pseudomonas aeruginosa (PaFd), Allochromatium vinosum (AlvinFd, Fig. C2) in order to correlate their structure to the widely different reduction potentials of their two metal clusters [–460 (cluster II) and –675 mV (cluster I)] [2,3]. The degree of exposure to the outer environment of cluster I sulfur atoms (Fig. C3) leads towards less negative reduction potentials (E°). This is better illustrated by V13G AlvinFd (high exposure, E° = 594 mV) and EcFd (low exposure, E° = 675 mV). In C57A AlvinFd (Fig. C3a), the movement of the protein backbone, as a result of replacing the non-coordinating Cys57 by Ala, leads to a +50 mV up-shift of the potential of the nearby cluster I, by removal of polar interactions involving the thiolate group and adjustment of the H-bonds network involving the cluster atoms.However, the accurate new structures we have determined for the mutated Alvin Fd, C57A and V13G (at 1.05 and 1.48 Å resolution [3], respectively) indicate the effects of subtle structural differences around cluster IIthat provide insight on the electrochemical properties. Namely, polar interactions of side chains and water molecules with the sulfur atoms of cluster II  (Fig. C2), which are absent in the environment of cluster I (the cluster with the lowest reduction potential), can be correlated to the ca. 180-250 mV difference between the reduction potentials of clusters I and II.

Fig. C2. The structure of the Alvin Fd variant, C57A (1.05 Å resolution, a). The environment of cluster II, although  buried inside the molecule, is comparable to the exposed cluster II of the widely studied clostridia ferredoxin (b) that has two isopotential clusters (ca. –400 mV) . This is due to a water molecule (W6) entrapped in the interior of the C57A that attracts side chains of polar residues close to the cluster II S-atoms. In contrast, cluster I of C57A is less polar than cluster II of AlvinFd and of clostridia ferredoxins.

Fig. C3Solvent-accessibility surfaces color-mapped according to the chemical nature of the surface atoms of a, C57A; b, V13G; c, PaFd; d,  EcFd: Nitrogen, oxygen, and carbon atoms are shown in blue, red, and green, respectively. The Cys11SG atoms of cluster I are shown in yellow at the central region of the surfaces. The exposed S1 atom of cluster I of V13G (b) is shown in magenta. The bulky sulfur atom of Met13 of EcFd (d), in orange, limits the exposure of the Cys11SG atom.

III.  Muscle proteins

Collaboration with the group of M. Wilmanns, EMBL-Hamburg on two muscle proteins titin and myomesin

 1.   The structure of the amino terminus titin/telethonin complex [4]: Titin (the largest protein made by human cells) along with myosin and actin, plays a crucial role in the muscle function. The structure of the 2:1 titin:telethonin complex gives the details of the interaction of two titin molecules with telethonin, a smallprotein-component of the the Z-disc of muscles (Fig. C4). This leads to a model of titin interaction in the Z-disc: two titin molecules from different sarcomeric filaments cross-linked at their amino terminus via telethonin. The study may lead to new insight for some muscle and cardiac diseases; moreover, it may provide a molecular paradigm about the cross-linking of major sarcomeric filaments.

Fig. C4. A: ribbon representation (in two orientations) of the two antiparallel titin immunoglobulin-like domains Z1 (blue, residues 1-98) and Z2 (cyan, residues 99-196 including the Z1-Z2 linker) cross-linked by telethonin (red) via extended antiparallel β-sheets involving the three molecules. B: surface representation of the titin-telethonin-titin complex, in two orientations (in green, the telethonin domain 60-90 not participating in β-sheets).

  2. The myomesin structure. The mechanical forces exerted by the muscles are sensed and buffered by unusually long and elastic filament proteins with highly repetitive domain structures. Myomesin is one such repetitive filament protein, which is thought to form bridges between the main contractile filament, that provides the muscle with resistance in the radial dimension. To investigate how the repetitive structure of myomesin contributes to muscle elasticity, the overall architecture has been determined  using a combination of four complementary structural biology methods [5].  The structure of the myomesin domains my9-my11 has been elucidated by X-ray crystallography. Each domain comprises a immunoglobulin-like and helix pattern (Fig. C5 in green). These have been combined with the already available structure of the final my12-my13 domains of the carboxyl terminus in order to make a model of how the myomesin filament attaches in the M-band of the muscle sarcomer.

Fig. C5. The complete myomesin (My) tail-to-tail filament structure: In ribbon representation the dimeric myomesin IgH domain array My9–My10–My11–My12–(My13)2–My12–My11’– My10’–My9′. Myomesin domains that have been structurally investigated are shown in violet (first molecule) and blue (second molecule). The helical linkers are shown in green. A ruler provides an overall length estimate of the filament.

IV.    Endoplasmic Reticulum Aminopeptidases

Endoplasmic Reticulum (ER) aminopeptidases ERAP1 and ERAP2 cooperate to trim a vast variety of antigenic peptide precursors to generate mature epitopes for binding onto MHC class I molecules and help regulate the adaptive immune response.  In collaboration with the group of E. Stratikos at our institution, whave determined for the 1st time the structure of ERAP2 to 3.08 Å by X-ray crystallography [6]. The ERAP2 structure (Fig. C6) provides a structural explanation for the different peptide N-terminus specificities between ERAP1 and ERAP2 and suggests that such differences extend throughout the whole peptide sequence. Overall, the structure helps explain how two homologous aminopeptidases cooperate to process a large variety of sequences, a key property to their biological role. Common coding single nucleotide polymorphisms (SNPs) in ERAP1 and ERAP2 have been linked with predisposition to human diseases ranging from viral and bacterial infections to autoimmunity and cancer. The common ERAP2 SNP rs2549782 that codes for amino acid variation N392K leads to alterations in both the activity and the specificity of the enzyme. Specifically, the 392N allele excises hydrophobic N-terminal residues from epitope precursors up to 165-fold faster compared to the 392K allele, although both alleles are very similar in excising positively charged N-terminal amino acids. This is primarily due to changes in the catalytic turnover rate (kcat) and not in the affinity for the substrate.  X-ray crystallographic analysis of the ERAP2 392K (Fig. C6)allele suggests that the polymorphism interferes with the stabilization of the N-terminus of the peptide both directly and indirectly through interactions with key residues participating in catalysis [7]. The study provides mechanistic insight to the association of this ERAP2 polymorphism with disease and support the idea that polymorphic variation in antigen processing enzymes constitutes a component of immune response variability in humans.

Fig. C6. Left: Ribbon representation of ERAP2  colored by domain (domain I in blue, II in green, III in orange and IV in magenta). Right: A,sequence alignment of ERAP2 and homologous aminopeptidases showing conservation of the polymorphic residue 392 (in bold); the adjacent aminopeptidase motif (HELAH) is also indicated; B, key catalytic residues in the ERAP2K structure are shown in stick representation and superimposed with equivalent residues in ERAP2N (2SE6). Note the relative positioning of Lys392 with respect to the catalytic Glu residues as well as the N-terminus of the bound ligand (Lysa). Electron density 2|Fo|-|Fcat 2σ is indicated around Lys392; C, pairwise electrostatic interaction energies (ΔΕinter in Kcal/mol) calculated between the polymorphic residue 392 and the Lysligand. The reported values are the average of the 20 runs with a standard deviation of less than 1%, which represents the uncertainty due to the granularity of the grid; D, superimposition of ERAP2 residues that cap the S1 specificity pocket of the enzyme. |Fo|-|Fc| electron density (at 2.5σcalculated in the absence of the ligand is shown around Lysa. 2|Fo|-|Fc| electron density at 2σ is indicated around Glu177.

V.    RNA Complexes

Structure determination of complexes of the ribosomal decoding aminoacyl site (A-site) of bacterial 16SrRNA constructs with synthetic analogs of aminoglycosides. Aminoglycoside antibiotics selectively bind to the A-site of bacterial 16SrRNA, interfering in the fidelity of proteosynthesis in the bacterial cellsThe designed compounds are synthesised at the collaborating organic Chemical Biology laboratory of our institution headed by Dr D. Vourloumis. Elucidation of the structure of the above compounds in complex with selective RNA constructs that are appropriate models of the A-site, provides the prerequisite for guiding this synthetic effort. Moreover, the structural information combined with biochemical and kinetic experiments is the first step in the development of pharmaceuticals for fighting bacterial infections by RNA-directed therapies.

Fig. C7. Model of the structure of a ligand-free bacterial RNA construct. Three consecutive molecules of RNA monomers (each represented by a different colour) form infinite continuous chains. The structure has been solved by the software IL MILIONE (Burla MC, Caliandro R, Camalli M, Carrozzini B, Cascarano GL, De Caro L, Giacovazzo C, Polidori G, Siliqi, D, Spagna R, J Appl CRYSTALLOG. 2007, 40   609-613)

VI. DsbD: a bacterial thiol-disulfide oxidoreductase

DsbD is one of the five proteins of the bacterial Disulfide bond (Dsb) formation system. It is located in the inner membrane of Gram-negative bacteria and it is responsible for transporting reducing power from the cytoplasm to the oxidising periplasm of the cell (Fig. C8). It comprises three domains; the central transmembrane domain (tmDsbD), of unknown structure, is flanked by two globular periplasmic domains, the N-terminal domain nDsbD) with an immunoglobulin-like fold and the C-terminal domain (cDsbD) with a classical thioredoxin (Trx) fold. DsbD plays an important role in oxidative protein folding because it allows for the correct formation of disulfide bonds in proteins functioning in harsh extracytoplasmic environments [8]. It is also involved in bacterial pathogenesis as the expression and stability of most virulent factors (secreted molecules, secretion apparatuses, adhesion systems etc) are dependent on the presence of DsbD. The structures of the N-terminal domain in the reduced form and of two point-mutants of the C-terminal domain have been determined [9,10] in collaboration with the Department of Biochemistry, University of Oxford (Prof. Stuart J. Ferguson, Prof. Christina Redfield and Dr Despoina A.I. Mavridou). This work contributed significantly in elucidating the interaction of the soluble domains of this unique oxidoreductase but also in the general understanding of the factors controlling the reactivity of the ubiquitous thioredoxin fold.

DsbD

Fig. C8. DsbD: The sole reductant provider in the periplasm.

VI. Macromolecular crystallization methods

New crystallisation methodology for biological macromolecules, in collaboration with Imperial CollegeLondon.

1. As Coordinators of the Industry-Academia Partnerships and Pathways – Marie Curie Project TOPCRYST, we developed the use of Dual Polarization Interferometry (Fig. C9), pioneered by Farfield Scientific Ltd., to probe crystallisation at its most crucial stages. This allows to predict the outcome of crystallisation trials when they are still at their earliest stages and thus to rationally design and direct such experiments in order to lead them to well-diffracting crystals [11]. Research on other techniques for effective a priori prediction of crystallisation conditions, such as the use of Genetic Algorithms and of calorimetry, is also being actively pursued [12].

DPI

Fig. C9. Optical principle of dual polarization interferometry. Light from a sensing waveguide in contact with the investigated solution interferes with that from a reference waveguide, resulting in interference fringes characterized by their phase and contrast.

2. Research into substances and materials promoting the heterogeneous nucleation of macromolecular crystals is a blossoming topic in crystallogenesis. We have participated in developing the use of materials containing pores of cavities, such as Bioglass, carbon nanotubes, and Molecularly Imprinted Polymers (Fig. C10), as heterogeneous nucleants and are pursuing further ideas [13-16].

crystals from nucleants

Fig. C10. Crystals of a protein (human macrophage Migration Inhibiting Factor) growing at metastable conditions in the presence of Molecularly Imprinted Polymer imprinted with protein

References